Atherosclerosis is a chronic inflammatory condition characterized by arterial plaques composed of cholesterol- filled macrophages, a necrotic lipid core, and a fibrous cap containing vascular smooth muscle cells (SMCs). Extensive work has defined how genetic variants altering lipid levels and inflammation contribute to atherosclerosis, but less is known as to how genetic alterations affecting SMCs predispose to atherosclerosis. An expanded role for SMCs in atherosclerotic lesions is evoked by the finding that SMCs in plaques lose SMC differentiation markers and initiate expression of macrophage markers, thus becoming a macrophage-like cell. Similarly, SMCs downregulate SMC markers in vitro, but upregulate macrophage markers with exposure to free cholesterol. We determined that some heterozygous missense mutations in ACTA2, which encodes the SMC-specific isoform of ?-actin, predispose individuals to both thoracic aortic disease and early onset coronary artery disease (CAD). We have engineered a mouse model with one such mutation and SMCs explanted from Acta2R149C/+ aortas de-differentiate and increase expression of macrophage markers at much lower concentrations of free cholesterol than wildtype SMCs. Furthermore, when the Acta2R149C/+ mice are crossed into Apoe-/- mice and fed a high fat diet, the double mutant mice have a significantly increased burden of atherosclerotic plaques when compared to similarly treated Apoe-/- mice. We hypothesize that early onset CAD associated with the SM ?-actin R149C mutation is due to disrupted folding of the mutant actin, leading to increased Klf4 activation and augmented SMC phenotypic switching to macrophage-like cells. We further speculate that, although many ACTA2 mutations may increase SMC proliferation and migration, only variants that increase SMC switching to macrophage-like cells will predispose to early onset CAD. The aims to address these hypotheses are the following: (1) Characterize the atherosclerotic lesions in the Acta2R149C/+ mice, including identifying the origin of the SMCs and macrophages in the lesions; (2) Identify cellular pathways responsible for enhanced Acta2R149C/+ SMC switching to a macrophage-like cell with exposure to cholesterol, and assess the role of these pathways in the increased plaque burden in Acta2R149C/+ mice through genetic manipulation. (3) Determine how CAD-associated ACTA2 mutations disrupt folding by the chaperonin CCT complex, and show causality between SM ?-actin folding defects and CAD-predisposing ACTA2 missense mutations. Thus, the proposed research will provide valuable new insights into the role of SMC phenotypic switching as a risk factor for atherosclerosis, and may also identify novel therapeutic targets that can delay or even prevent disease progression.